IMPACT™

IMPACT™ (Intein Mediated Purification with an Affinity Chitin-binding Tag) is a novel protein purification system which utilizes the inducible self-cleavage activity of a protein splicing element (termed intein) to separate the target protein from the affinity tag. It distinguishes itself from all other purification systems by its ability to purify, in a single chromatographic step, a native recombinant protein without the use of a protease (1,2). A target protein is fused to a tag consisting of the intein and the chitin binding domain which allows affinity purification of the fusion protein on a chitin column. In the presence of thiols such as DTT or 2-mercaptoethanesulfonic acid (MESNA), the intein undergoes specific self-cleavage which releases the target protein from the chitin-bound intein tag resulting in a single-column purification of the target protein. In addition, with the use of pTXB1, native recombinant proteins that possess a reactive C-terminal thioester can be isolated for applications including protein ligation, semisynthesis and site-specific labeling (3,4).

The IMPACT™ Kit (NEB #E6901) contains expression vectors which allow fusion of the cleavable intein tag to either the C-terminus or N-terminus of the target protein. This flexibility in fusion protein construction maximizes the probability of successful expression and purification of a target protein.

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